Evaluation of the Immunity Responses in Mice to Recombinant Bacillus subtilis Displaying Newcastle Disease Virus HN Protein Truncations

Bacillus subtilis, a probiotic bacterium with engineering potential, is widely used for the expression of exogenous proteins. In this study, we utilized the integrative plasmid pDG364 to integrate the hemagglutinin–neuraminidase (HN) gene from Newcastle disease virus (NDV) into the genome of the B. subtilis 168 model strain. We successfully constructed a recombinant B. subtilis strain (designated B. subtilis RH) that displays a truncated HN antigen fragment on the surface of its spores and further evaluated its immunogenic effects in mice. Using ELISA, we quantified the levels of IgG in serum and secretory IgA (sIgA) in intestinal contents. The results revealed that the recombinant B. subtilis RH elicited robust specific mucosal and humoral immune responses in mice. Furthermore, B. subtilis RH demonstrated potential mucosal immune adjuvant properties by fostering the development of immune organs and augmenting the number of lymphocytes in the small intestinal villi. Additionally, the strain significantly upregulated the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestinal mucosa. In conclusion, the B. subtilis RH strain developed in this study exhibits promising mucosal immunogenic effects. It holds potential as a candidate for an anti-NDV mucosal subunit vaccine and offers a novel preventive strategy for the poultry industry against this disease.


Introduction
Bacillus subtilis is a probiotic bacterium that is widely used for engineering purposes to express exogenous proteins [1,2].Under conditions of extreme stress or nutrient scarcity, B. subtilis generates a distinct cellular form known as the spore.The spore's core and cortex are crucial for its formation and integrity.Encompassing the cortex is the spore's coat layer, composed of approximately 80 unique coat proteins, which collectively define the spore surface [3].This robust architecture endows B. subtilis spores with high resistance, enabling them to withstand acid and bile salt challenges within the intestine tract [4,5].Beyond its capacity to elicit both mucosal and humoral immune responses within the host, B. subtilis also functions as an effective carrier or adjuvant for mucosal vaccine delivery [6].Leveraging spore surface display technology enables the stable presentation of antigenic proteins on the spore surface.Research efforts have yielded the development of protective antigens through the utilization of diverse coat proteins, such as cotB, cotC, cotG, etc., as anchoring agents.This approach facilitates the expression of antigenic proteins on the spore surface and culminates in the creation of recombinant probiotics that are rich in immunogenic properties [7][8][9].Recombinant strains of B. subtilis are characterized by their ease of storage and transport, which simplifies the immunization process and reduces stress in animals.These strains exhibit a broad spectrum of applications in the field of animal immunization.Recent advances have led to the successful display of various antigenic proteins on the spore surface, including tetanus toxoid fragment C, ovalbumin [10,11], cholera toxin [12], the transmissible gastroenteritis virus spike protein [13], the highly pathogenic avian influenza H5N1 hemagglutinin protein [14], and the Vibrio OmpK protein [15].The induction of specific immune responses by these recombinant probiotics underscores their exceptional immune attributes and probiotic functionalities.
Newcastle disease virus (NDV) is the causative agent of Newcastle disease (ND), a significant poultry disease.Characterized by high contagiousness, ND is categorized by the World Organization for Animal Health (WOAH) as a List A disease in animals, necessitating legal reporting [16].In China, the Ministry of Agriculture ranks it as a Class II animal epidemic disease.Challenges in eradicating NDV stem from suboptimal vaccination, antigenic drift, limited duration of immunity, and immunosuppressive conditions [17][18][19].The hemagglutinin-neuraminidase (HN) protein, a surface glycoprotein of NDV, is renowned for its ability to elicit the production of neutralizing antibodies within poultry, thereby serving as a pivotal protective antigen.The globular head domain of the HN protein houses the essential functional regions, including the active site and all antigenic sites [20,21].This domain is instrumental in the virus's infectivity and pathogenicity, positioning the HN protein as an optimal target for the genetic engineering of vaccines aimed at combating ND [22][23][24].Currently, the main types of genetically engineered vaccines for ND include nucleic acid vaccines, subunit vaccines, and live-vector vaccines [25][26][27].The attachment and entry of NDV into host cells are facilitated by its binding to two major salic acid (SA) receptors present on the membrane of target cells.Specifically, SAα2,3-Gal receptors are prevalent in chicken small intestinal epithelial cells, while SAα2,6-Gal receptors are more common in avian trachea ciliated epithelial cells [28,29].Consequently, NDV exhibits a specific mucosal tropism and can readily infect birds through the respiratory and digestive tracts.Mucosal vaccines have the capacity to elicit the production of sIgA, establishing mucosal immunity through various immunization routes such as intranasal, ocular, drinking water, and aerosol administration.These methods significantly enhance local immunity within the respiratory and digestive tracts [30,31].In summary, mucosal immunization plays a crucial role in managing ND, and the development of recombinant probiotic-based mucosal vaccines against NDV is currently a paramount strategy in this field.
In this study, we engineered probiotics using the B. subtilis model strain 168.By integrating a fusion gene into the genome of B. subtilis 168 via the integration plasmid pDG364, we successfully constructed the recombinant B. subtilis RH.This strain displays the truncated HN (HNJD) protein of Newcastle disease virus on its spore surface.We then proceeded to assess the specific immune response and mucosal immune adjuvant effect elicited by B. subtilis RH in mice.The novel oral vaccine developed herein offers an innovative approach for the prevention of NDV-related diseases, presenting a fresh alternative in disease management strategies.

Strains, Vaccines, Cell, Plasmids, Primers Sequences, and Experimental Ethics
The bacterial strains (Escherichia coli BL21/DH5α and Bacillus subtilis 168), vaccines (NDV vaccine strain LaSota/CS2), chick embryo fibroblasts (CEF), plasmids (pUCm-T, pET-32a, and pDG364-cotB), and primer sequences utilized in this study are itemized in Supplementary Table S1.All methodologies and animal experimentation were carried out in accordance with the "Guide for the Care and Use of Laboratory Animals", receiving approval from the Institutional Animal Care and Use Committee of Sichuan Agricultural University (approval number: SYXK Chuan 2019-187).

Cloning of Truncated HN Gene
The E.Z.N.A ® Total RNA Kit II (Omega, Honolulu, HI, USA) was used to extract the total viral RNA from the NDV LaSota vaccine strain (GenBank accession No. AY845400.2).Subsequently, the extracted RNA was reverse-transcribed to synthesize cDNA.The hemagglutininneuraminidase-truncated (HNJD) gene was PCR-amplified using the cDNA as a template and HNJD-F1/R1 primers [32].The amplicon was then T-A-cloned into a pUCm-T vector to generate the recombinant plasmid pUCm-T-HNJD, which was sequenced and subjected to site-directed mutagenesis to eliminate potential restriction sites that might interfere with the subsequent steps (performed by Tsingke Biotechnology Co., Ltd., Beijing, China).

Preparation of Hyperimmune Serum and Prokaryotic Expression of HNJD
Female New Zealand rabbits weighing 2.0 ± 0.2 kg were used to produce anti-NDV hyperimmune serum.Each rabbit received a subcutaneous injection of 2 mL of the live vaccine (LaSota strain).This was followed by weekly booster immunizations, and serum collection occurred subsequent to three consecutive vaccinations.Hemagglutination inhibition (HI) assays were performed to determine the HI antibody titers against NDV, and optimal dilutions were ascertained and preserved for further use.
The pUCm-T-HNJD plasmid and the pET-32a vector were digested with BamH I and EcoR I enzymes (Takara Bio, Dalian, China).The resulting target fragments were ligated to form the expression plasmid pET-32a-HNJD1, which was then transformed into E. coli BL21.The successfully expressing strain was designated as E. coli BL21/pET-32a-HNJD.The expression of the recombinant protein was induced with isopropyl-β-D-thiogalactoside (IPTG) added to a Luria-Bertani (LB) broth at a final concentration of 1 mM.After incubation at 37 • C with shaking at 160 rpm for 6 h, 1 mL of bacterial culture was collected, while the pET-32a vector alone served as a negative control.The bacterial cells were lysed by ultrasonication and centrifuged at 10,000× g for 1 min.The pellet was collected and subjected to SDS-PAGE, followed by transfer onto a PVDF membrane (Solarbio, Beijing, China).The membrane was blocked with 5% skim milk at 37 • C for 2 h and then incubated overnight at 4 • C with rabbit-derived NDV hyperimmune serum (diluted 1:200 in TBST).HRP-conjugated goat anti-rabbit IgG (diluted 1:2000 in TBST, BOSTER, Wuhan, China) was used as the secondary antibody.After a 2 h incubation at room temperature and washing with TBST, color development was performed using the DAB Color Development Kit (BOSTER, Wuhan, China) according to the manufacturer's instructions.If the HNJD protein specifically binds to anti-NDV hyperimmune serum, a brown band should be visible on the membrane after immunoblotting.

Construction and Transformation of Recombinant Integration Plasmid
The plasmid pDG364-cotB developed in our previous work [33], was utilized.Employing the methodology detailed in Section 2.3, we constructed the recombinant plasmid pDG364-cotB-HNJD.Competent cells of B. subtilis 168 were prepared following Julkowska et al.'s protocol [34].The plasmid pDG364-cotB-HNJD was linearized with an Xba I enzyme digestion prior to being introduced into the competent cells.Through homologous doublecrossover recombination, the target gene was integrated into the amyE (amylase) locus of B. subtilis (Figure 1B).Positive clones were identified on chloramphenicol (5 µg/mL) resistance plates and further screened on a 1% starch nutrient agar medium.Genomic DNA from the recombinant bacteria was extracted, and a PCR analysis was performed with primer pairs amyE-F/R, amyE-F/HNJD-R2, cotB-F/HNJD-R2, and HNJD-F2/R2.Amplification products were verified by gel electrophoresis.The correctly identified recombinant strain was designated B. subtilis RH.
F2/R2.Amplification products were verified by gel electrophoresis.The correctly identified recombinant strain was designated B. subtilis RH.

Sporulation and Immunofluorescence Microscopy
Sporulation was induced using Difco sporulation medium (DSM) according to the method described by Stasiłojć et al. [35].After approximately 24 h of sporulating culture, spores were treated with 2.0 mg/L of lysozyme at 37 °C for 2 h to eliminate any residual vegetative cells.The purified spores were then obtained by sequential washing with 1 M of NaCl, 1 M of MgCl2, and distilled water, as detailed in reference [7].Finally, the spread plate technique was employed to enumerate the spore colonies.
Immunofluorescence microscopy was used to verify the successful display of the HNJD protein on the spore surface [36], as indicated in Figure 1C.A purified spore suspension was prepared and affixed to microscope slides according to the method described previously [37].Rabbit anti-NDV hyperimmune serum (diluted 1:200 in PBST) served as the primary antibody, while Cy3-conjugated goat anti-rabbit IgG (diluted 1:200 in PBST, BOSTER, Wuhan, China) was used as the secondary antibody.Additionally, serum from non-immunized mice was applied as the primary antibody to establish a negative control.The immunofluorescence images were captured using a fluorescence microscope (DMi8, Leica, Tokyo, Japan).

Sporulation and Immunofluorescence Microscopy
Sporulation was induced using Difco sporulation medium (DSM) according to the method described by Stasiłojć et al. [35].After approximately 24 h of sporulating culture, spores were treated with 2.0 mg/L of lysozyme at 37 • C for 2 h to eliminate any residual vegetative cells.The purified spores were then obtained by sequential washing with 1 M of NaCl, 1 M of MgCl 2 , and distilled water, as detailed in reference [7].Finally, the spread plate technique was employed to enumerate the spore colonies.
Immunofluorescence microscopy was used to verify the successful display of the HNJD protein on the spore surface [36], as indicated in Figure 1C.A purified spore suspension was prepared and affixed to microscope slides according to the method described previously [37].Rabbit anti-NDV hyperimmune serum (diluted 1:200 in PBST) served as the primary antibody, while Cy3-conjugated goat anti-rabbit IgG (diluted 1:200 in PBST, BOSTER, Wuhan, China) was used as the secondary antibody.Additionally, serum from non-immunized mice was applied as the primary antibody to establish a negative control.The immunofluorescence images were captured using a fluorescence microscope (DMi8, Leica, Tokyo, Japan).

Immunization of Mice and Collection of Samples
A total of 100 three-week-old female BALB/c mice underwent a seven-day acclimatization period before being randomly distributed into five groups, each comprising 20 individuals.Each group was then subdivided into five cages, with four mice per cage.The feeding regimen and immunization schedule for the mice are detailed in Figure 2. The mice in group A served as the control group and were provided with a basal diet.Group B received a diet mixed with 2.0 × 10 6 CFU per gram of wild-type B. subtilis 168 spores (168-spores).Group C was intraperitoneally administered 100 µL of inactivated vaccine (strain LaSota, with viral content equal to or exceeding 1.0 × 10 8 ELD50 prior to inactivation) on days 1, 15, and 29.Group D was fed a diet containing 2.0 × 10 6 CFU per gram of B. subtilis RH spores (RH-spores).Group E received an RH-spore suspension (2.0 × 10 10 CFU/mL) via oral gavage.The mice in group E received 0.1 mL daily on days 1-3, 15-17, and 29-31, as previously described [38].Each mouse was given 3-5 g of this diet daily.
The experimental period spanned 42 days.On days 0, 14, 28, and 42, five mice from each group were randomly selected, and their serum and small intestinal contents were harvested and preserved at −80 °C.The body weight of the mice was recorded on day 42.Following euthanasia, ileal tissues and contents were gathered, and the ileal tissues were preserved in 4% paraformaldehyde.The spleens and thymuses were excised, and organ indices were calculated as follows: organ index = weight of organ (mg)/weight of body(g) Figure 2. Schematic representation of the vaccination and feeding trail.Group A: untreated control; Group B: mixed diet with 168-spores; Group C: inactivated vaccine administered; Group D: mixed diet with RH-spores; Group E: RH-spores administered via gavage.

Detection of NDV-Specific Serum and Mucosal Antibodies
Serum IgG antibody levels were quantified using a competitive enzyme-linked immunosorbent assay (ELISA) with the Serum Antibodies to Newcastle Disease Virus ELISA Kit (Zhenrui Bio, Shenzhen, China).The serum IgG levels were expressed as S/N (sampleto-negative control) values: / = (OD450 nm of sample well) (OD450 nm of negative control well) ⁄ The indirect ELISA method was used to detect the anti-NDV sIgA in the small intestinal contents.The ELISA plates were coated with NDV antigen, provided by Shenzhen The experimental period spanned 42 days.On days 0, 14, 28, and 42, five mice from each group were randomly selected, and their serum and small intestinal contents were harvested and preserved at −80 • C. The body weight of the mice was recorded on day 42.Following euthanasia, ileal tissues and contents were gathered, and the ileal tissues were preserved in 4% paraformaldehyde.The spleens and thymuses were excised, and organ indices were calculated as follows: organ index = weight of organ (mg)/weight of body (g)

Detection of NDV-Specific Serum and Mucosal Antibodies
Serum IgG antibody levels were quantified using a competitive enzyme-linked immunosorbent assay (ELISA) with the Serum Antibodies to Newcastle Disease Virus ELISA Kit (Zhenrui Bio, Shenzhen, China).The serum IgG levels were expressed as S/N (sampleto-negative control) values: S/N = (OD450 nm of sample well)/(OD450 nm of negative control well) The indirect ELISA method was used to detect the anti-NDV sIgA in the small intestinal contents.The ELISA plates were coated with NDV antigen, provided by Shenzhen Zhenrui Biotech Co., Ltd., Shenzhen, China.The intestinal contents were diluted to 1:100 with PBST, and serum from unimmunized mice served as the negative control.The secondary antibody used was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgA (diluted to 1:1200 in PBST, Abcam, Cambridge, UK).The levels of sIgA in the small intestinal contents were expressed as P/N (positive-to-negative control) values: P/N = (OD450 nm of sample well)/(OD450 nm of negative control well)

Microneutralization Test
The serum-neutralizing antibody (NA) titers were detected with a microneutralization assay, as previously described [39].CEF cells were seeded in 96-well plates and used for virus neutralization tests when they reached a confluency rate of 50-60%.All serum samples underwent heat inactivation at 56 • C for 30 min and were then serially diluted two-fold from 1:50 to 1:1600 in Dulbecco's Modified Eagle Medium (DMEM).Subsequently, 50 µL of the NDV CS2 vaccine strain (200 TCID50) was combined with an equal volume of the diluted serum and incubated together at 37 • C for 1 h.This mixture was then added to a 96-well plate containing CEF cells, with three replicate wells for each sample.The cells were maintained in DMEM supplemented with 2% fetal bovine serum (FBS) at 37 • C and 5% CO 2 .It is crucial to note that if the virus is neutralized by serum antibodies, it will be unable to infect the cells.After incubation for 72 h at 37 • C and 5% CO 2 , the NA titer for each group was calculated as the geometric mean titer (GMT), which was established by identifying the lowest serum dilution that prevented cytopathic effects in over 50% of the CEF cells.

Hemagglutination Inhibition Test
The HI assay was performed in accordance with the National Standard of the People's Republic of China [40].Serum samples were twofold diluted in a 96-well plate.A total of 25 µL of NDV antigen containing four hemagglutination units was introduced into each well, including a positive control.After 30 min of incubation, 25 µL of a 1% suspension of chicken red blood cells was added to each well and incubated for an additional 30 min.The results were observed, and the HI titers were defined as the highest dilution of serum that completely inhibited NDV hemagglutination.

Gene Expression of Cytokine in Ileum
Total RNA from ileal tissues samples was extracted using the Animal Total Isolation Kit (Foregene Co., Ltd., Chengdu, China).The extracted RNA was subsequently reversetranscribed into cDNA using the RT-Easy™ II (Foregene, Chengdu, China).The expression levels of cytokine-related mRNAs, including interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), were quantified by real-time quantitative PCR (RT-qPCR) according to the methodology described by Xin et al. [41].Primer sequences are listed in Supplementary Table S2.Betaactin (β-actin) served as the reference gene for data normalization.The relative expression of the target genes was calculate using the 2 −∆∆CT method:

The Morphology of Ileum
Tissue sections were prepared from ileal tissue fixed in 4% paraformaldehyde (Service-Bio Technology Co., Ltd., Wuhan, China).The imaging of the target areas was performed using an Eclipse Ci-L photographic microscope (Nikon, Tokyo, Japan), following the method described previously [42].Image analysis was conducted using Image-Pro Plus 6.0 software, with results reported in millimeters.For each intestinal sample, five random fields were selected to measure the villus height (VH) and crypt depth (CD) and to determine the VH/CD ratio.A 100× magnification field was used, and five intact villi were chosen from each tissue section for measurement.The height of the intestinal villi and the number of intraepithelial lymphocytes (IELs) were measured.The number of IELs per unit length of villus was then calculated as follows: Number of IELs per unit height = IELs/height of intestinal villus 2.12.Statistical Analysis Data were analyzed using one-way ANOVA followed by the Friedman test for multiple comparisons.Statistical analyses were conducted with IBM SPSS Statistics 26 (IBM Corporation, Armonk, NY, USA), and results are presented as mean ± standard deviation (SD).Statistical significance was denoted by letters, where different letters indicate significant differences (p < 0.05) between groups.Similar letters indicate no significant difference among groups (p > 0.05).Data visualization was performed using Origin 2021 (OriginLab, Northampton, MA, USA).

Construction and Transformation of Recombinant Plasmid
Figure 3 illustrates the construction of recombinant plasmids and the outcomes of the transformation.The HNJD gene was approximately 642 bp in size (Figure 3A).Sequencing revealed that the HNJD gene contained an EcoR I restriction site, with the original and mutated sequences detailed in Supplementary Table S3.The HI titer of the rabbit anti-NDV hyperimmune serum reached a value of 13log2.An analysis via 12% SDS-PAGE and Western blotting (shown in Figure 3B,C) confirmed that the E. coli BL21/pET-32a-HNJD expression product exhibited a protein band around 42 kDa, signifying successful recombinant protein expression with reactivity to NDV-specific antibodies.Following double-enzyme digestion validation, the recombinant plasmid pDG364-cotB-NHJD was introduced into B. subtilis-competent cells (Figure 3D).PCR identification was performed using four primer sets, with the wild-type B. subtilis genome serving as the control.The results presented in Figure 3E demonstrated that all DNA bands were congruent with their theoretical sizes.Furthermore, Figure 3F indicated that the recombinant B. subtilis lost its ability to break down starch due to the insertion of the fusion gene cotB-HNJD into the amylase-encoding locus.In conclusion, we successfully engineered a strain of B. subtilis RH.
random fields were selected to measure the villus height (VH) and crypt depth (CD) and to determine the VH/CD ratio.A 100× magnification field was used, and five intact villi were chosen from each tissue section for measurement.The height of the intestinal villi and the number of intraepithelial lymphocytes (IELs) were measured.The number of IELs per unit length of villus was then calculated as follows: Number of IELs per unit height = IELs height of intestinal villus ⁄

Statistical Analysis
Data were analyzed using one-way ANOVA followed by the Friedman test for multiple comparisons.Statistical analyses were conducted with IBM SPSS Statistics 26 (IBM Corporation, Armonk, NY, USA), and results are presented as mean ± standard deviation (SD).Statistical significance was denoted by letters, where different letters indicate significant differences (p < 0.05) between groups.Similar letters indicate no significant difference among groups (p > 0.05).Data visualization was performed using Origin 2021 (OriginLab, Northampton, MA, USA).

Construction and Transformation of Recombinant Plasmid
Figure 3 illustrates the construction of recombinant plasmids and the outcomes of the transformation.The HNJD gene was approximately 642 bp in size (Figure 3A).Sequencing revealed that the HNJD gene contained an EcoR I restriction site, with the original and mutated sequences detailed in Supplementary Table S3.The HI titer of the rabbit anti-NDV hyperimmune serum reached a value of 13log2.An analysis via 12% SDS-PAGE and Western blotting (shown in Figure 3B,C) confirmed that the E. coli BL21/pET-32a-HNJD expression product exhibited a protein band around 42 kDa, signifying successful recombinant protein expression with reactivity to NDV-specific antibodies.Following doubleenzyme digestion validation, the recombinant plasmid pDG364-cotB-NHJD was introduced into B. subtilis-competent cells (Figure 3D).PCR identification was performed using four primer sets, with the wild-type B. subtilis genome serving as the control.The results presented in Figure 3E demonstrated that all DNA bands were congruent with their theoretical sizes.Furthermore, Figure 3F indicated that the recombinant B. subtilis lost its ability to break down starch due to the insertion of the fusion gene cotB-HNJD into the amylase-encoding locus.In conclusion, we successfully engineered a strain of B. subtilis RH.

Sporulation and Immunofluorescence Microscopy
The results of the indirect immunofluorescence assay for the recombinant B. subtilis RH are presented in Figure 4. Red fluorescent signals were exclusively observed in B. subtilis RH when viewed under a microscope, using anti-NDV hyperimmune serum as the primary antibody.This observation confirms that the HNJD protein was successfully expressed on the surface of B. subtilis RH spores and was capable of binding to NDV antibodies.

Sporulation and Immunofluorescence Microscopy
The results of the indirect immunofluorescence assay for the recombinant B. subtilis RH are presented in Figure 4. Red fluorescent signals were exclusively observed in B. subtilis RH when viewed under a microscope, using anti-NDV hyperimmune serum as the primary antibody.This observation confirms that the HNJD protein was successfully expressed on the surface of B. subtilis RH spores and was capable of binding to NDV antibodies.

Detection of NDV-Specific Serum and Mucosal Antibodies
The ELISA results for serum IgG and mucosal sIgA are depicted in Figure 5.The serum IgG levels were quantified using a competitive ELISA approach.The S/N value is inversely related to the concentration of IgG, with lower S/N values indicating higher IgG

Detection of NDV-Specific Serum and Mucosal Antibodies
The ELISA results for serum IgG and mucosal sIgA are depicted in Figure 5.The serum IgG levels were quantified using a competitive ELISA approach.The S/N value is inversely related to the concentration of IgG, with lower S/N values indicating higher IgG concentrations.Throughout the study, Groups A and B exhibited consistently lower IgG levels, whereas Groups C, D, and E had significantly elevated serum IgG levels (p < 0.05), with Group C demonstrating the highest concentration of serum IgG.The serum IgG level was higher in Group D compared to Group E. The sIgA levels in the ileal content were determined using an indirect ELISA method.Higher P/N values are indicative of increased sIgA levels.It was observed that Groups A, B, and C did not generate substantial levels of sIgA antibodies.In contrast, both Groups D and E showed a marked increase in sIgA levels (p < 0.05), which progressively rose over time, reaching a plateau on day 28.Notably, Group D achieved higher levels of sIgA than Group E. This suggests that B. subtilis RH effectively triggered an immune response in the mice through both mixed feeding and gavage administration methods.The mixed feeding method elicited a stronger mucosal immune response, potentially due to an extended period of stimulation of the mucous membranes.concentrations.Throughout the study, Groups A and B exhibited consistently lower IgG levels, whereas Groups C, D, and E had significantly elevated serum IgG levels (p < 0.05), with Group C demonstrating the highest concentration of serum IgG.The serum IgG level was higher in Group D compared to Group E. The sIgA levels in the ileal content were determined using an indirect ELISA method.Higher P/N values are indicative of increased sIgA levels.It was observed that Groups A, B, and C did not generate substantial levels of sIgA antibodies.In contrast, both Groups D and E showed a marked increase in sIgA levels (p < 0.05), which progressively rose over time, reaching a plateau on day 28.Notably, Group D achieved higher levels of sIgA than Group E. This suggests that B. subtilis RH effectively triggered an immune response in the mice through both mixed feeding and gavage administration methods.The mixed feeding method elicited a stronger mucosal immune response, potentially due to an extended period of stimulation of the mucous membranes.

Detection of NA and HI Antibody Titers
Using the Reed-Muench method, the TCID50 for the NDV CS2 vaccine strain was determined to be 10 −6.2 /0.1 mL.As depicted in Figure 6, Groups C, D, and E exhibited significantly elevated NA and HI titers compared to Groups A and B (p < 0.05).Group C achieved the highest NA and HI titers, and Group D exhibited significantly higher HI titers than Group E (p < 0.05).These findings indicate that mucosal immunization can elicit the robust production of neutralizing antibodies in mice and that the levels of NA and HI antibodies are correlated with the route of mucosal administration.

Detection of NA and HI Antibody Titers
Using the Reed-Muench method, the TCID50 for the NDV CS2 vaccine strain was determined to be 10 −6.2 /0.1 mL.As depicted in Figure 6, Groups C, D, and E exhibited significantly elevated NA and HI titers compared to Groups A and B (p < 0.05).Group C achieved the highest NA and HI titers, and Group D exhibited significantly higher HI titers than Group E (p < 0.05).These findings indicate that mucosal immunization can elicit the robust production of neutralizing antibodies in mice and that the levels of NA and HI antibodies are correlated with the route of mucosal administration.concentrations.Throughout the study, Groups A and B exhibited consistently lower IgG levels, whereas Groups C, D, and E had significantly elevated serum IgG levels (p < 0.05), with Group C demonstrating the highest concentration of serum IgG.The serum IgG level was higher in Group D compared to Group E. The sIgA levels in the ileal content were determined using an indirect ELISA method.Higher P/N values are indicative of increased sIgA levels.It was observed that Groups A, B, and C did not generate substantial levels of sIgA antibodies.In contrast, both Groups D and E showed a marked increase in sIgA levels (p < 0.05), which progressively rose over time, reaching a plateau on day 28.Notably, Group D achieved higher levels of sIgA than Group E. This suggests that B. subtilis RH effectively triggered an immune response in the mice through both mixed feeding and gavage administration methods.The mixed feeding method elicited a stronger mucosal immune response, potentially due to an extended period of stimulation of the mucous membranes.

Detection of NA and HI Antibody Titers
Using the Reed-Muench method, the TCID50 for the NDV CS2 vaccine strain was determined to be 10 −6.2 /0.1 mL.As depicted in Figure 6, Groups C, D, and E exhibited significantly elevated NA and HI titers compared to Groups A and B (p < 0.05).Group C achieved the highest NA and HI titers, and Group D exhibited significantly higher HI titers than Group E (p < 0.05).These findings indicate that mucosal immunization can elicit the robust production of neutralizing antibodies in mice and that the levels of NA and HI antibodies are correlated with the route of mucosal administration.

Gene Expression of Cytokine in the Ileum
Cytokines related to immunity were quantified using RT-qPCR, and the results are shown in Figure 7.We observed a significant upregulation in the relative mRNA expression of IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the spore-immunized groups (Group B, D, and E).Notably, all cytokine mRNA expression levels were higher in Group D compared to Group E (p < 0.05), indicating that mixed feeding was more effective than gavage administration.While the expression of IL-6 and IL-10 was significantly elevated in Group C compared to Group A (p < 0.05), their levels remained lower than those in the spore-immunized groups.This indicates that inactivated vaccines can enhance cytokine expression in the intestinal mucosa by modulating the systemic immune response, but this effect is less pronounced than the direct action of the spores on the mucosal surface.

Gene Expression of Cytokine in the Ileum
Cytokines related to immunity were quantified using RT-qPCR, and the results are shown in Figure 7.We observed a significant upregulation in the relative mRNA expression of IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the spore-immunized groups (Group B, D, and E).Notably, all cytokine mRNA expression levels were higher in Group D compared to Group E (p < 0.05), indicating that mixed feeding was more effective than gavage administration.While the expression of IL-6 and IL-10 was significantly elevated in Group C compared to Group A (p < 0.05), their levels remained lower than those in the sporeimmunized groups.This indicates that inactivated vaccines can enhance cytokine expression in the intestinal mucosa by modulating the systemic immune response, but this effect is less pronounced than the direct action of the spores on the mucosal surface.Expression levels of cytokines IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestine were quantified using RT-qPCR.The data were normalized using the −2 ΔΔCT method.The results are presented as mean ± SD, with a sample size of n = 5.Statistical analysis was conducted using oneway ANOVA, followed by a Friedman test.Bars labeled with distinct lowercase letters denote a statistically significant difference at the p < 0.05 level.

Histomorphology and Intraepithelial Lymphocytes of the Mouse Ileum
Figure 8A illustrates the ileum morphology of the mice from each group, revealing a clear and intact histological structure of the ileum.The measurements of VH and CD in the mouse ileum are shown in Figure 8B.The mice from the spore-immunized groups exhibited a significant increase in VH and the VH/CD ratio and a decrease in CD compared to Groups A and C (p < 0.05).However, no significant differences were observed among the spore-immunized groups (p > 0.05).The number of IELs per unit length of the intestinal villi was quantified and is also presented in Figure 8B.The spore-immunized groups exhibited a significantly higher number of IELs per unit length compared to Groups A and C (p < 0.05), with Group D demonstrating the highest count.These findings suggest that the administration of B. subtilis 168 and RH can effectively enhance intestinal mucosa development and promote the maturation of intestinal structures in mice.Expression levels of cytokines IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestine were quantified using RT-qPCR.The data were normalized using the −2 ∆∆CT method.The results are presented as mean ± SD, with a sample size of n = 5.Statistical analysis was conducted using one-way ANOVA, followed by a Friedman test.Bars labeled with distinct lowercase letters denote a statistically significant difference at the p < 0.05 level.

Histomorphology and Intraepithelial Lymphocytes of the Mouse Ileum
Figure 8A illustrates the ileum morphology of the mice from each group, revealing a clear and intact histological structure of the ileum.The measurements of VH and CD in the mouse ileum are shown in Figure 8B.The mice from the spore-immunized groups exhibited a significant increase in VH and the VH/CD ratio and a decrease in CD compared to Groups A and C (p < 0.05).However, no significant differences were observed among the spore-immunized groups (p > 0.05).The number of IELs per unit length of the intestinal villi was quantified and is also presented in Figure 8B.The spore-immunized groups exhibited a significantly higher number of IELs per unit length compared to Groups A and C (p < 0.05), with Group D demonstrating the highest count.These findings suggest that the administration of B. subtilis 168 and RH can effectively enhance intestinal mucosa development and promote the maturation of intestinal structures in mice.Notably, B. subtilis RH appears to be more effective in stimulating the proliferation of IELs and modulating the immune response within the intestinal mucosa.
Notably, B. subtilis RH appears to be more effective in stimulating the proliferation of IELs and modulating the immune response within the intestinal mucosa.

Mouse Immune Organ Index
Figure 9 demonstrates the spleen index was increased in all groups except for Group A. Notably, Group D exhibited a significantly higher spleen index than both Group C and Group E (p < 0.05).In contrast, no significant differences were found in the thymus index across all the groups (p > 0.05).

Mouse Immune Organ Index
Figure 9 demonstrates that the spleen index was increased in all groups except for Group A. Notably, Group D exhibited a significantly higher spleen index than both Group C and Group E (p < 0.05).In contrast, no significant differences were found in the thymus index across all the groups (p > 0.05).

Mouse Immune Organ Index
Figure 9 demonstrates that the spleen index was increased in all groups except for Group A. Notably, Group D exhibited a significantly higher spleen index than both Group C and Group E (p < 0.05).In contrast, no significant differences were found in the thymus index across all the groups (p > 0.05).

Discussion
In the realm of livestock and poultry production, probiotics are increasingly being adopted as a viable alternative to antibiotics [43].B. subtilis, a probiotic species, has been acknowledged by the FDA as safe and is commonly incorporated into functional feed additives due to its robust probiotic characteristics and the convenience of its isolation and cultivation.This bacterium exhibits remarkable resistance to the harsh conditions within the gastrointestinal tract, enabling it to maintain potent biological activity in the digestive system [44,45].It contributes to enhanced digestion by providing enzymes, antimicrobial peptides, and other advantageous metabolic byproducts, which collectively improve feed efficiency, growth performance, and the integrity of the gut barrier [46,47].B. subtilis also plays a pivotal role in modulating the intestinal microbiota and the immune response, thereby warding off pathogenic microorganisms and preventing diseases [48][49][50].In addition, B. subtilis is often employed as an engineering bacterium for the efficient expression of exogenous proteins.A technique for displaying proteins on the surface of B. spores was first proposed by Isticato et al. [51] in 2001.They successfully expressed a 459-amino-acid C-terminal fragment of the tetanus toxin (TTFC) on a spore surface, demonstrating its reactivity.With the elucidation of the B. subtilis genome and advances in proteomic research, this technique has been effectively utilized across various domains, including mucosal immunization, industrial enzyme production, environmental remediation, pharmaceutical development, and more [3].Owing to its probiotic attributes and stability, B. subtilis holds promising advantages as a mucosal adjuvant for the delivery of antigenic proteins.Recombinant probiotics displaying the HN protein can elicit specific immune responses when used as mucosal vaccines, offering substantial potential in research and application areas.Our laboratory has successfully constructed several recombinant B. subtilis strains that display heterologous proteins, including Salmonella OmpC [52], Rotavirus VP8 [33], and Porcine circovirus type 2 Cap proteins [53].In the current study, we excised a portion of the antigenic region from the HN protein and spliced it into smaller fragments following Chen's methodology [32].This approach preserved the native hemagglutinin-neuraminidase active sites of the HN protein, along with the majority of its antigenic sites and all the cysteine residues crucial for its tertiary structure.The resulting truncated HN protein is efficiently expressed in prokaryotic systems as a monomer, in contrast to the wild-type tetramer form.The prokaryotically expressed HNJD protein is readily recognized by NDV-specific antibodies, demonstrating robust reactivity.Immunofluorescence assays corroborated the accurate expression and correct anchoring of the HNJD protein to the spore surface during sporulation.
Upon ingestion by animals, B. subtilis spores can safely navigate through the stomach and germinate within the upper intestine, where they undergo a brief period of multiplication before sporulating again in the lower intestine [54,55].Both spores and vegetative cells possess the capacity to interact with intestinal epithelial cells, thereby stimulating the proliferation of intestinal-associated lymphocytes and fortifying the mucosal immune system [56].The mucosal immune system is pivotal for immune defense, acting as the body's initial barrier against pathogenic invasion.In the context of preventive vaccination against Newcastle disease, eliciting an effective mucosal immune response can significantly diminish the rates of infection and morbidity, constituting the most efficacious strategy for the prevention and control of the disease [57].In our investigation, we discovered that B. subtilis RH has the capacity to induce both systemic and local mucosal immune responses.It was observed that supplementing mice with B. subtilis RH resulted in elevated levels of serum IgG and intestinal sIgA.sIgA is the most abundant immunoglobulin produced by the intestinal mucosa, contributing to mucosal homeostasis by inhibiting pathogen adherence to the mucosal surface and playing a vital role in the early prevention of infection [58].In contrast, commercial vaccines administered via intraperitoneal injection fail to stimulate the production of anti-NDV-specific sIgA in mice.This limitation is due to the fact that most commercial vaccines delivered through non-intestinal routes provide only partial protection against clinical infections and do not completely eliminate infection at the site of initial mucosa invasion.Chicks immunized with commercially inactivated vaccines often require mucosal immune adjuvants to elicit the effective production of specific mucosal antibodies, as indicated in previous studies [59,60].HI and NA assays are used to measure antibody potency, reflecting their capacity to neutralize the virus and prevent infection.Through microneutralization and HI assays, we have shown that serum antibodies derived from mice vaccinated with RH-spores effectively neutralize NDV, preventing its cellular invasion.This suggests that antigen delivery via spores can effectively penetrate the selectively permeable mucus layer of the intestinal tract, leading to the generation of antibodies and the secretion of cytokines by associated lymphocytes [61,62], which contributes to combating disease infections.Gonçalves et al. [15] immunized zebrafish with spores displaying the Vibrio antigen OmpK, leading to a 50-90% increase in survival rates after infection with Vibrio anguillarum and Vibrio parahaemolyticus.Oh et al. [63] constructed a recombinant B. subtilis strain expressing the protective antigen (PA) of Bacillus anthracis, which successfully elicited a protective immune response in mice, with the level of protection varying according to the immunization method used.In our study, we observed that the immunogenic effect was dependent on the mode of administration, with the mixed feeding group showing superior performance over the intermittent gavage group in all measured parameters, including IgG and sIgA levels, as well as NA and HI titers.This could be attributed to the fact that dietary spores germinate in the jejunum and ileum, whereas spores and vegetative cells do not permanently colonize the intestine and are only in it for a brief duration [54,64].Our findings suggest that continuous feeding immunization is more effective at inducing intestinal mucosal immunity and probiotic effects than high-dose gavage administered a limited number of times, aligning with previous research conducted in our laboratory.Additionally, gavage may induce stress and disrupt homeostasis in animals, indicating that intermittent gavage is not an effective or suitable immunization method.
In our study, B. subtilis RH modulated the expression levels of cytokines such as IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in mouse ileal tissues.These cytokines are instrumental in orchestrating the Th1/Th2 pathway, which is essential for the regulation of both innate and adaptive immune responses in animals [65].CD4 + T lymphocytes can differentiate into either Th1 or Th2 cell subsets.The elevated expression of IL-1β, TNF-α, and IFN-γ contributes to cellular immunity by fostering a converging Th1-type immune response.IFN-γ, acting as a pivotal activator of cellular immunity, stimulates CD8 + T lymphocytes and macrophages to eliminate foreign pathogens or infected cells.On the other hand, IL-6 and IL-10 respond to Th2 chemotaxis and also propel the proliferation of T and B cells, thereby enhancing the innate immune response [35].Unlike many commercially inactivated vaccines that predominantly elicit Th2-type responses [66], B. subtilis RH is capable of promoting both cellular and innate immunity.The spores of the wild-type B. subtilis strain also upregulated cytokine expression, demonstrating their capacity to bolster the immune system.This observation aligns with the findings reported by LEE et al. [67].
Compared to conventional injectable vaccines, mucosal vaccines offer convenience, enhanced safety, and cost-effectiveness.Our investigation has demonstrated that B. subtilis RH can elicit a specific immune response when administered orally, highlighting its potential as an adjuvant for mucosal vaccines, which is particularly advantageous in the livestock and poultry industries.Measures such as gut histomorphometry and immune organ indices are frequently employed in research to assess gastrointestinal development and performance.The morphology of the small intestine directly mirrors the health of the gut, as well as its digestive and absorptive capabilities, which are critical for sustaining the intestinal immune system [68][69][70].Longer villi correlate with epithelial cell proliferation and can stimulate cell mitosis, whereas shorter villi and deeper crypts are associated with nutrient malabsorption and diminished gastrointestinal function [71][72][73][74].Our research has determined that dietary supplementation with B. subtilis RH results in an increase in the height of ileal villi and a decrease in crypt depth, thereby enhancing intestinal tissue structure in mice.In related findings, Dong et al. [75] found that B. subtilis BYS2 not only increased the height of avian duodenal and jejunal villi but also increased the villi-to-crypts rates, reduced viral load following NDV infection, and significantly improved survival rates.Jayaraman et al. [76] discovered that B. subtilis PB6 could replace antibiotic growth promoters (AGP) to prevent necrotizing enterocolitis while concurrently improving small intestine organization and the growth performance of broilers through mixed feeding.The intestinal mucosa's epithelial layer is replete with IELs, which are crucial for preserving intestinal epithelial integrity and play a pivotal role in immune surveillance and cell-mediated mucosal immunity [56,77].In our study, both strains of B. subtilis effectively stimulated the proliferation of IELs in the mouse ileum, thereby enhancing immunoprotection against virus challenges and potentially blocking the infection route of NDV.However, it remains uncertain if this effect is replicated with commercial vaccines.The organ index serves as an objective measure to assess the growth and development of immune organs and is a crucial parameter for evaluating the micro-ecological impacts of probiotics [78,79].Our findings indicate that B. subtilis RH promotes splenic growth in mice, yet its influence on thymic development was not evident on day 42.This could be attributed to the fact that the thymus is among the first immune organs to develop and undergoes age-related involution [80], culminating in no significant disparity between the experimental groups.Furthermore, B. subtilis RH has been utilized as a genetically engineered probiotic for animal feed at the recommended dosage (1.0-2.0 × 10 6 CFU/g), which is consistent with that of most probiotics [81,82].This approach ensures that it functions as an immune adjuvant while eliciting an effective immune response.In a related study by Pham et al. [83], when the conventional dose (1.0 × 10 6 CFU/g) of CotB-VP28 spores in feed pellets was increased by 50-fold (5.0 × 10 7 CFU/g) or 1000-fold (1.0 × 10 9 CFU/g) for black tiger shrimps, the enhancement in protection against spot syndrome virus infection was marginally increased by only 2.5% and 7.5%, respectively.Thus, routine dosages of spore supplementation can provide effective immune protection, and higher concentrations are not only unnecessary but also impractical in the poultry industry due to cost implications.Nonetheless, we posit that the optimal concentration of various recombinant B. subtilis strains may differ depending on the vectors used, the expression methods, and efficiency.Future poultry experiments could explore the immunoprotective effects across different dosages.In summary, these findings indicate that B. subtilis RH, when used as a feed additive, significantly enhances the development of intestinal and immune organs in animals, promotes the proliferation of immune-related lymphocytes, and shows promise as a mucosal immune adjuvant.
Although B. subtilis RH was capable of inducing an effective specific immune response, the IgG levels and NA titers were not as high as those achieved with commercial vaccines.One plausible explanation for this discrepancy could be the absence of a linker peptide, which might impact the expression of exogenous proteins.The inclusion of a linker peptide can form a stable helical structure that bridges the anchor protein and the target protein, alleviating rigidity issues and enhancing their stability and expression.However, the direct fusion of anchor proteins may lead to undesirable outcomes such as incorrect folding, low yields, and compromised biological activity [84].Hinc et al. [85] compared the expression efficiency of fusion proteins in cotZ-UreA versus cotB-GGGEAAAKGGGG-UreA, finding that the latter, which incorporates flexible linker peptides, expressed up to 100-fold more recombinant proteins on a spore than the former.Although various types of linker peptides have been designed over time [86,87], the ideal configuration remains elusive.The selection of different anchor proteins [8,85] and ligation strategies [88,89] can also significantly enhance the presentation efficiency of exogenous proteins.Determining how to improve the display efficiency of the HNJD protein on the spore surface may be a focus of our future research endeavors.Looking ahead, omics technologies should be harnessed to uncover the precise mechanisms through which B. subtilis RH induces immune responses and exerts its probiotic effects.

Conclusions
In this study, we developed a recombinant B. subtilis RH strain capable of expressing the NDV HN protein on the surface of its spores.This innovation induced both mucosal and systemic immune responses in mice.Additionally, the recombinant B. subtilis enhanced the expression of cytokines in the ileum, fostered the development of immune organs and the intestinal tract, and stimulated the proliferation of intestinal-associated lymphocytes.As such, it also serves as a mucosal immune adjuvant, positioning it as a potential novel probiotic strain engineered to combat NDV.The evaluation of its immunogenicity in mice conducted herein lays the groundwork for future challenge studies in poultry models.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/microorganisms12030439/s1.The supplementary files provided include Table S1, which lists the trains, vaccines, cell lines, plasmids, and primer sequences used; Table S2, detailing the primer sequences for real-time quantitative PCR; and Table S3, presenting the HNJD gene sequences along with the optimized sites.

Figure 1 .
Figure 1.Schematic representation of homologous recombination and the structure of RH spores.(A) Diagram of the integrative plasmid pDG364-cotB-HNJD.(B) Homologous double-crossover recombination between the recombinant plasmid pDG364-cotB-HNJD and the genome of B. subtilis 168 occurs, leading to the integration of the target gene into the amyE locus.(C) Structure depiction of RH spores showing the fusion expression of the anchor protein cotB and the HNJD protein on the surface of the spores.

Figure 1 .
Figure 1.Schematic representation of homologous recombination and the structure of RH spores.(A) Diagram of the integrative plasmid pDG364-cotB-HNJD.(B) Homologous double-crossover recombination between the recombinant plasmid pDG364-cotB-HNJD and the genome of B. subtilis 168 occurs, leading to the integration of the target gene into the amyE locus.(C) Structure depiction of RH spores showing the fusion expression of the anchor protein cotB and the HNJD protein on the surface of the spores.

Figure 2 .
Figure 2. Schematic representation of the vaccination and feeding trail.Group A: untreated control; Group B: mixed diet with 168-spores; Group C: inactivated vaccine administered; Group D: mixed diet with RH-spores; Group E: RH-spores administered via gavage.

Figure 4 .
Figure 4. Indirect immunofluorescence assay.(A) Spores of B. subtilis 168 and RH observed under 40× magnification using bright-field microscopy; (B) Spores of B. subtilis 168 and RH under 40× magnification using fluorescence microscopy, with different serums serving as the primary antibody and Cy3-conjugated goat anti-rabbit IgG employed as the secondary antibody.

Figure 4 .
Figure 4. Indirect immunofluorescence assay.(A) Spores of B. subtilis 168 and RH observed under 40× magnification using bright-field microscopy; (B) Spores of B. subtilis 168 and RH under 40× magnification using fluorescence microscopy, with different serums serving as the primary antibody and Cy3-conjugated goat anti-rabbit IgG employed as the secondary antibody.

Figure 5 .
Figure 5.Immune responses in mice following immunization using different methods.Serum IgG levels are depicted as S/N values, while sIgA levels in the mucosa are represented by P/N values.The data are reported as mean values with SD, based on a sample size of n = 5.Statistical analysis involved one-way ANOVA, followed by a Friedman test.Bars labeled with distinct lowercase letters indicate a significant difference at the p < 0.05 level.

Figure 5 .
Figure 5.Immune responses in mice following immunization using different methods.Serum IgG levels are depicted as S/N values, while sIgA levels in the mucosa are represented by P/N values.The data are reported as mean values with SD, based on a sample size of n = 5.Statistical analysis involved one-way ANOVA, followed by a Friedman test.Bars labeled with distinct lowercase letters indicate a significant difference at the p < 0.05 level.

Figure 5 .
Figure 5.Immune responses in mice following immunization using different methods.Serum IgG levels are depicted as S/N values, while sIgA levels in the mucosa are represented by P/N values.The data are reported as mean values with SD, based on a sample size of n = 5.Statistical analysis involved one-way ANOVA, followed by a Friedman test.Bars labeled with distinct lowercase letters indicate a significant difference at the p < 0.05 level.

Figure 6 .
Figure 6.Determination of NA and HI levels in the serum of each group.The data are presented as mean ± SD, with a sample size of n = 5.Statistical analysis was conducted using one-way ANOVA,

Figure 7 .
Figure 7. Expression levels of cytokines IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestine were quantified using RT-qPCR.The data were normalized using the −2 ΔΔCT method.The results are presented as mean ± SD, with a sample size of n = 5.Statistical analysis was conducted using oneway ANOVA, followed by a Friedman test.Bars labeled with distinct lowercase letters denote a statistically significant difference at the p < 0.05 level.

Figure 7 .
Figure 7. Expression levels of cytokines IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in the small intestine were quantified using RT-qPCR.The data were normalized using the −2 ∆∆CT method.The results are presented as mean ± SD, with a sample size of n = 5.Statistical analysis was conducted using one-way ANOVA, followed by a Friedman test.Bars labeled with distinct lowercase letters denote a statistically significant difference at the p < 0.05 level.

Figure 8 .
Figure 8. Histomorphologic analysis and enumeration of IELs in ileal tissue.(A) Representative images of ileal tissue from each group are shown, with intestinal crypt (1) and villi (2) indicated by black arrows.IELs are localized within the mucosal epithelial layer of the small intestine, characterized by their irregularly shaped nuclei and darker staining relative to the surrounding epithelium.Microscopic magnification is set at 100×.(B) Quantitative measurements of intestinal CD, VH, VH/CD ratio, and IELs per unit length are presented.Data are presented as the mean ± SD, with a sample size of n = 5.Statistical analysis involved one-way ANOVA, followed by a Friedman test, where bars labeled with distinct lowercase letters signify a significant difference at the p < 0.05 level.VH refers to the height of the villus, CD denotes the depth of the crypts, and the VH/CD ratio represents the metric of the villus height relative to the crypt depth.IELs are defined as intestinal intraepithelial lymphocytes.

Figure 8 .
Figure 8. Histomorphologic analysis and enumeration of IELs in ileal tissue.(A) Representative images of ileal tissue from each group are shown, with intestinal crypt (1) and villi (2) indicated by black arrows.IELs are localized within the mucosal epithelial layer of the small intestine, characterized by their irregularly shaped nuclei and darker staining relative to the surrounding epithelium.Microscopic magnification is set at 100×.(B) Quantitative measurements of intestinal CD, VH, VH/CD ratio, and IELs per unit length are presented.Data are presented as the mean ± SD, with a sample size of n = 5.Statistical analysis involved one-way ANOVA, followed by a Friedman test, where bars labeled with distinct lowercase letters signify a significant difference at the p < 0.05 level.VH refers to the height of the villus, CD denotes the depth of the crypts, and the VH/CD ratio represents the metric of the villus height relative to the crypt depth.IELs are defined as intestinal intraepithelial lymphocytes.

Microorganisms 2024 ,
12, x FOR PEER REVIEW 11 of 19Notably, B. subtilis RH appears to be more effective in stimulating the proliferation of IELs and modulating the immune response within the intestinal mucosa.

Figure 8 .
Figure 8. Histomorphologic analysis and enumeration of IELs in ileal tissue.(A) Representative images of ileal tissue from each group are shown, with intestinal crypt (1) and villi (2) indicated by black arrows.IELs are localized within the mucosal epithelial layer of the small intestine, characterized by their irregularly shaped nuclei and darker staining relative to the surrounding epithelium.Microscopic magnification is set at 100×.(B) Quantitative measurements of intestinal CD, VH, VH/CD ratio, and IELs per unit length are presented.Data are presented as the mean ± SD, with a sample size of n = 5.Statistical analysis involved one-way ANOVA, followed by a Friedman test, where bars labeled with distinct lowercase letters signify a significant difference at the p < 0.05 level.VH refers to the height of the villus, CD denotes the depth of the crypts, and the VH/CD ratio represents the metric of the villus height relative to the crypt depth.IELs are defined as intestinal intraepithelial lymphocytes.

Figure 9 .
Figure 9. Organ indices of the thymus and spleen.Data are presented as mean ± SD, with a sample sized of n = 5.Statistical analysis was conducted using one-way ANOVA, followed by a Friedman test.Bars labeled with different lowercase letters indicate a statistically significant difference at the p < 0.05 level.

Author
Contributions: J.L. and M.Y. contributed equally to this study.J.L., M.Y. and K.P. contributed to the conceptualization; the methodology was developed by J.L., M.Y., B.C., Z.W., Y.C. and K.P.; the formal analysis was conducted by J.L. and M.Y.; the resources were provided by D.Z. and Y.Z.; data curation involved B.C., Z.W. and Y.C.; the original draft was written by J.L. and M.Y.; X.N., Y.Z. and K.P. reviewed and edited the draft; visualization was created by Y.Y. and M.Z.; supervision was provided by X.N., Y.Z. and K.P.; project administration was managed by K.P.; funding acquisition was secured by K.P.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Technology Innovation Research Team in the University of Sichuan Province (Grant No. KM406183.1),the Sichuan Province Science and Technology Plan Key Research and Development Project (Grant No. 2021FYS0002), the Sichuan Agricultural University Double Branch Program (Grant No. 03571146), and the Chengdu Agricultural College Science and Technology Fund (Grant No. 20QN01).
Determination of NA and HI levels in the serum of each group.The data are presented as mean ± SD, with a sample size of n = 5.Statistical analysis was conducted using one-way ANOVA, followed by a Friedman test.Bars labeled with different lowercase letters indicate a statistically significant difference at the p < 0.05 level.